an extra PCR-band just below... - (Oct/09/2006 )
I'm expecting an amplicon of 1200 bp, and so there is...but there is an extra band just below, can't tell how much smaller but they are very close. The upper band is 'the big one', the lower many times weaker.
I've tried to increase the extension time from 1 min to 2, didn't help, neither increasing the temperature.
So, any tips??
It could be primer dimer - you may need to redesign your primers (wost case), but playing with the MgCl concentration could help.
try annaeling gradient from 60 to 70 degrees, if this doesnt help do a MgCl gradient: use a range from 1.5mM to 3mM you might see an improvement.
if not check you primer design. or you could start introducing DMSO but i think things just start getting messy if you do.
If it's a primer dimer it should be much smaller ~50 bp but it could be if you ran your gel for a very short time. It's probably just a non-specific band, I would try decreasing the primer concentration (I usually use 0.2 uM final in a 25 ul reaction). I always use primers with 50% GC content and 63 degrees annealing temp works well for me. You could also try using a hot start taq if you're not, as this helps to decrease non-specific bands in my experience.
Thanx for the tips!
It can't be PD's, I didn't mentioned but the size is 1200 bp..
I'm using 0,15 µM primer, so that not so much. I've ordered new primers now, waiting for them, if they don't solve the problem I'll test Mg concentration.